Novel 3-(pyrazol-4-yl)-2-(1H-indole-3-carbonyl)acrylonitrile derivatives induce intrinsic and extrinsic apoptotic death mediated P53 in HCT116 colon carcinoma

A novel series of α-cyano indolylchalcones was prepared, and their chemical structures were confirmed based on the different spectral data. Among them, compound 7f was observed to be the most effective bioactive chalcone with distinguished potency and selectivity against colorectal carcinoma (HCT116) with IC50 value (6.76 µg/mL) relative to the positive control (5 FU) (77.15 µg/mL). In a preliminary action study, the acrylonitrile chalcone 7f was found to enhance apoptotic action via different mechanisms like inhibition of some anti-apoptotic protein expression, regulation of some apoptotic proteins, production of caspases, and cell cycle arrest. All mechanisms suggested that compound 7f could act as a professional chemotherapeutic agent. Also, a molecular docking study was achieved on some selected proteins implicated in cancer (Caspase 9, XIAP, P53 mutant Y220C, and MDM2) which showed variable interactions with compound 7f with good Gibbs free energy scores.


Antiproliferative effect of α-cyano indolylchalcones
The prepared compounds 7a-f were screened against two cancer cell lines, lung carcinoma (A549), and colon cancer (HCT116).Also, the compounds were tested against the normal human skin melanocyte cell line (HFB4) for the sake of comparison.As indicated in Table 1 and Fig. 3, the cytotoxic effect of our synthesized series against HCT116 was better than the A549 cell line.The IC 50 values ranged from 6.76 to 202.08 µg/mL for HCT116, while they ranged from 193.93 to 1,014.00 µg/mL for A549.Concerning the HCT116 cell line, it was found that compound 7f was the most promising compound (IC 50 = 6.76 µg/mL) compared to 5 fluorouracil (positive control) (IC 50 = 77.15µg/mL).Compound 7d exerted a good cytotoxic effect (IC 50 = 43 µg/mL) relative to the positive control, while the IC 50 value for compound 7a was 93.1 µg/mL which means moderate activity.The remaining compounds 7b, 7c, and 7e showed weak cytotoxic activity (IC 50 = 187.81, 202.08, and 195.41 µg/mL, respectively).Concerning A549, all the tested compounds showed lower IC 50 values than the positive control (IC 50 = 371.36µg/mL) except for compound 7e which exerted a very weak effect (Table 1).Among them, compound 7f showed the lowest IC 50 value (193.93µg/mL)relative to 5fluorouracil.While, compounds 7a, 7b, 7cand 7d showed comparable IC 50 values (208.58, 238.14, 274.60, and 269 µg/mL, respectively).From the results of the cytotoxicity against normal HFB4 cells (Table 1), it was noticed that compound 7f was the most selective toward HCT116 (SI = 8.4), (Selectivity index (SI) = IC 50 in normal cells/IC 50 in tumor cells).

Structure-activity relationship
The structure-activity relationship of the prepared compounds toward HCT116 can be regarded in view of the substitution on the arly group at position-3 of the pyrazole ring.In the case of X = NO 2 as in compound 7f (Fig. 4), the cytotoxic activity and selectivity increased toward HCT116 as compared to the other compounds.It was noticed that compound 7d (X = OH) had better anticancer activity than the positive control and was selective  toward HCT116 cells.When X = H as in compound 7a, the activity was found to be moderate.For compounds 7b, 7c, and 7e (X = CH 3 , OCH 3 , and Cl) respectively, the activity was lower than that of the standard.Concerning A549, the activity of all compounds of the series except compound 7e (X = Cl) was better than the positive control and the best of them was compound 7f (X = NO 2 ).But the selectivity of the prepared compounds deteriorated against A549.

Gene expression analysis
In this current research, relative gene expression related to apoptosis should be studied well because they were very interesting to understand the effect of our selected compound on this important biological process.HCT 116 cells were treated with chalcone 7f (6.76µg/mL) for 48 h, and then subjected to qPCR analysis regarding control www.nature.com/scientificreports/samples.The effect of chalcone 7f on the expression level of (Bax, p53, Bcl2, and CDK4) genes was assessed using the qPCR technique.As illustrated in Table 2 and Fig. 5, the results indicated that mRNA expression values of tested apoptotic (Bax and p53) and anti-apoptotic (Bcl2 and CDK4) genes outlined promising and interesting effects regarding negative control.As indicated in this schematic diagram, the expression of apoptotic genes (Bax and P53) was enhanced by the effect of chalcone 7f to reach (3.662, and 5.435) respectively, relative to the control.Also, from the resulting plot, the anti-apoptotic genes (bcl2 and CDK4) were down-regulated effectively to values (0.351 and 0.454) respectively, by 6.76µg/mL of chalcone 7f in comparison with negative control.

Effect of chalcone 7f on caspase 3 production in HCT116
Apoptosis is known and identified by specific characterizations in which the activation of caspases plays an important role.In apoptotic pathways, mechanisms of (activation/inactivation) of these proteins are not fully The lead compound composed of three main moieties; Indole, pyrazole, and acrylonitrile.
In case of the substituent X= NO 2 , the activity was enhanced against HCT116 and A549 comparing to the other substituents.HN   understood.The significance of understanding the mechanistic machinery that controls programmed cell death, being initiated by various chemotherapeutic agents is very critical and attracts the attention of researchers in this area.In this assay, cells were incubated in the absence or presence of 7f with varying concentrations (39, 78,  156, 313, 625, 1250, and 2500 ng/ml), inducing a concentration-dependent increase in caspase3 production in HCT116.The maximal increase in caspase 3 production occurred with 7f concentrations between 1250 ng/ml and 2500 ng/ml as illustrated in Fig. 6.A concentration of 7f was used at its IC 50 value and compared directly to the control.Incubation of 7f with HCT116 resulted in high production of 504.1 pg/ml of caspase 3 protein.
However, there was only 103.5 pg/ml induced by control cells.It was interesting to note that chalcone 7f stimulated the production of caspase 3 protein in HCT116 cells 4.8-fold compared to control as outlined in Fig. 7.

Effect of chalcone 7f on caspase 8 production in HCT116
The production of caspase 8 was determined using various concentrations of 7f in a concertation-dependent manner.There was a gradual increase in the caspase 8 production level using the following concentrations (0.156, 0.312, 0.625, 1.25, 2.5, 5, and 10 ng/ml).The maximal increase in caspase 8 production was achieved by using 7f concentrations between 5 and 10 ng/ml (Fig. 8).HCT116 cells treated with 7f resulted in a 3.788-fold increase in caspase 8 production level.It achieved 1.104 ng/ml of caspase 8 compared to the untreated HCT116 cell line which was able to produce only 0.2914 ng/ml of caspase 8 (Fig. 9).www.nature.com/scientificreports/

Effect of 7f compound on caspase 9 production in HCT116
The compound 7f induced caspase 9 production in a dose-response approach.This was achieved by using the following concentrations of chalcone 7f (1.6, 3.1, 6.3, 12.5, 25, 50, 100 ng/ml).There was a continuing rise in the caspase 9 production level by increasing the concentrations regularly.A concentration between 50 and 100 ng/ml induced a maximum caspase 9 production as shown in Fig. 10.Compound 7f was able to stimulate the production of caspase 9 by inducing a 21.43 ng/ml of caspase 9.This showed about a 4.641-fold increase compared to control cells (Fig. 11).

Flow cytometer analysis
Chalcone 7f was selected for more investigations, in attempts to find its molecular action on cancer cells.It illustrated high and very interesting cytotoxicity toward the tested colon cancer cell line (HCT116) as mentioned in the above MTT assay data (Table 1).A flow cytometric report was utilized in this recent paper to investigate the effect of our novel acrylonitrile compound 7f on the cell cycle, HCT 116 cells were treated with compound 7f at a dose of (6.76 µg/mL) for 48 h and subjected to flow cytometric analysis.The data presented in Figs. 12 and 13 obviously indicated that chalcone 7f caused cell-growth arrest at G1/S phase relative to the untreated control HCT 116 cell line.At 6.76 µg/mL, compound 7f showed (51.33%) cell accumulation in the G1/S phase at the expense of other phases especially the mitotic phase G2/M phase (2.55%).Also, it was clearly appeared that chalcone 7f stimulated apoptotic induction of colon cancer cells significantly to reach 21.38% compared to control cells (0.32%) as shown in Figs. 14 and 15.    www.nature.com/scientificreports/

DNA fragmentation
Both control and treated cells were incubated for 48h.Then cells of both two samples were harvested for DNA fragmentation measurements using a SIGMA DNA fragmentation imaging kit with catalogue number 06432344001.To verify the validity of the DNA of both control and cells treated with 6.76 µg/mL of chalcone 7f, we carried out the loading of all samples on 2% agarose gel containing 1 µg/mL ethidium bromide and visualized by ultraviolet transillumination.As indicated in Fig. 16, a duplicate run for each sample was performed.It was illustrated that chalcone 7f completely deteriorated the genomic DNA of colorectal carcinoma after 48 h of treatment with respect to the control sample.The finding was promising and surprising and suggested that chalcone 7f was a promising chemotherapeutic agent.

Molecular docking study on compound 7f
The interactions of compound 7f with the crystal structure of caspase 9, X-linked inhibitor of apoptosis protein (XIAP), P53 cancer mutant Y220C, and Mouse double minute 2 homolog (MDM2) proteins (PDB code: 1jxq, 3eyl, 5o1h and 4wt2, respectively) were examined using molecular Operating Environment and BIOVIA Discovery Studio programs.As shown in Table 3, the RMSD values ≤ 2 (2, 0.86, 0.7, and 2 respectively) which demonstrated the high accuracy of our molecular docking results.Also, it was reported from Table 3 that the binding energies for compound 7f were − 21.8, − 16.5, − 20.1, and − 24.7 kcal/mol respectively, which were comparable to that of the co-crystallized standard ligands (− 33.7, − 24.9, − 15.8, and − 33.9 kcal/mol respectively).Figure 17a showed that compound 7f interacted with caspase 9 through 12 interactions, one hydrogen bond between the nitrogen of pyrazole moiety and ARG: 341 with bond distance 4.97 Å, another hydrogen bond between the oxygen of carbonyl group and LYS: 290 with bond distance 5.38 Å, two π-sulfur electrostatic interactions between the indole ring and CYS: 285, six π-cation electrostatic interactions with ARG: 177, ARG: 341, HIS: 237, CYS: 285 and LYS: 290, π-π T-shaped hydrophobic interaction between the indole ring and TRP: 340, and π -alkyl  hydrophobic interaction between the indole ring and VAL: 338.Regarding XIAP, compound 7f was bound with 6 interactions (Fig. 17b).These interactions were assigned as follows, a hydrogen bond between NH of indole ring and ASP: 309 (4.51 Å), π-donor hydrogen bond between benzene and LEU: 307 (4.52 Å), π-cation electrostatic interaction between phenyl ring and LYS: 297, π-π T-shaped and π-alkyl hydrophobic interactions between the benzene ring and, TRP: 323 and LEU: 307 respectively, and electrostatic interaction between the nitrogen of the nitro group and GLU: 314.While P53 cancer mutant Y220C was activated via 8 interactions.It was shown from Fig. 17c that these interactions included a carbon-hydrogen bond with ASP: 148 with bond distance 3.81 Å, π-donor hydrogen bond between the pyrazole ring and THR: 150 with bond distance 5.74 Å, three π-cation electrostatic interactions with ARG: 110 and ASP: 148 and three π-alkyl hydrophobic interactions with PRO: 223.At last, 7 interactions were seen between MDM2 and compound 7f (Fig. 17d).Among them, one hydrogen bond between the nitrogen of pyrazole ring and HIS: 96 (5.92 Å), one π-alkyl hydrophobic interaction between phenyl moiety and LYS: 94, one π-alkyl interaction between pyrazole moiety and VAL: 93, one π-alkyl interaction between indole ring and ILE: 61, one electrostatic interaction between the benzene ring and HIS: 96, and two electrostatic interactions between nitro group and HIS: 96.It was noticed that the moieties that involved in the interactions with the selected proteins were pyrazole, indole, and p-nitrobenzene.It is worth mentioning that the p-nitrobenzene substituent played a great role in the binding to the active sites of the tested proteins.

Discussion
The lead compound of our series is composed of indole, pyrazole, and acrylonitrile moieties.The diversity of the prepared compounds was based on the substituent (X) as mentioned before in the structure-activity relationship section.It was reported from the literature the promising biological activities of indole, pyrazole, and acrylonitrile which encouraged us to synthesize a series of compounds that combined all these moieties 51,61,62 .In our previous study 63 , It was found that 3-(thiophen-2-yl)pyrazol-4-yl)chalcone exerted a promising cytotoxic effect against HepG2 and A549 with IC 50 values of 64 and 66.6 µM, respectively.Another example for pyrazolyl chalcone was 1-[3,5-dimethyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-3-(p-tolyl) prop-2-en-1-one which showed promising anticancer activity against A549 and HepG2 with IC 50 equalled 44.3 and 57.9 µg/cm 3 , respectively 64 .Regarding indolyl chalcones, Jumaah et al., found that (E)-3-(6-methoxy-1-methyl-1H-indol-3-yl)-1-(3,4,5-trimethoxyphenyl)-prop-2-en-1-one, displayed high cytotoxic activities (IC 50 = 19.18µM) against MCF-7 65 .Kumar et al. 66 suggested that the trans geometry of the molecule resulting from α-substitution may be responsible for the enhanced cytotoxic effects over unsubstituted counterparts.Lawrence et al. 67 also studied the improved cytotoxic effects of the α-cyano substituent group on α,β-unsaturated enone system.The current study revealed that chalcone 7f was the most effective compound against HCT116 (IC 50 = 6.76 µg/mL) compared to 5-fluorouracil (IC 50 = 77.15µg/mL).Regarding qPCR data, as depicted in Table 2, our findings showed that chalcone 7f effectively up-regulated the apoptotic BAX gene with a fold change value of 3.662.Also, it effectively increased the expression level of the P53 gene with a fold change of 5.435.On the other hand, chalcone 7f strongly downregulated gene expression of anti-apoptotic genes, bcl2, and CDK4 with fold change values of 0.351 and 0.454, respectively.It was observed the high expression value of the tumour suppressor p53 gene (5.435)relative to the control confirmed the promising cytotoxic effect of chalcone 7f in the enhancement of the apoptotic death of colon cancer cells mediated p53 mechanism.This current data was very compatible with our previous literature that tested derivatives similar to our current target chalcone against colon and breast carcinoma respectively 68,69 .
Most of the studies broadly emphasized the apoptotic effect of chalcone compounds as anticancer agents 70,71 .
Similarly, another study verified that chalcone derivatives are considered promising antitumor mediators as illustrated in earlier literature suggested that chalcones can stimulate apoptosis 72,73 .The involvement of chalcone compounds in increasing the production of caspases has been evaluated previously by several studies using diverse carcinoma cells.For instance, Mohamed et al. 74 agreed with this recent study and showed that heterocyclic cyanoacrylamide compounds such as p-fluorophenyl and p-phenolic compounds had an effect on caspases production in liver carcinoma cells (Hepg2) through increasing the activity of caspases-3, -8, and -9.Additional evidence for the interesting results outlined in this recent paper has also been reported by Syamet al. 70 that showed apoptosis induction in breast cancer (MCF7) through stimulating the production of caspase-7, caspase-8, and caspase-9 using synthesized chalcone derivatives.Also, it was reported the ability of chalcones to induce apoptosis in HCT116 cells 68 .Therefore, this was confirmed in the present study where our compound 7f was capable of stimulating apoptosis in HCT116 cells by producing caspases-3, -8, and -9 in a concentration-dependent manner as demonstrated in Figs. 6, 8, and 10, respectively.It is well established that caspases play a crucial role during the death of proteases as they are a family of cysteine-dependent aspartate-directed proteases.These caspases are classified into two main classes: upstream (initiator) caspases and downstream (effector) caspases.Upstream caspases involve caspase-8 and caspase-9 which can cleave inactive pro-forms of downstream caspases such as caspase-3 and caspase-7 that cleave proteins contributed in processes of cell 51,75 .Furthermore, almost all studies approved that apoptosis can occur through two main pathways: the extrinsic (at the plasma membrane) and the intrinsic pathways (at the mitochondria) 76 .The activation of caspases-3 and -9 is elicited via the intrinsic pathway, whereas caspase-3 and -8 are activated through extrinsic pathway 74 .Therefore, we could conclude that our chalcone 7f stimulated the apoptotic pathway through both intrinsic and extrinsic mechanisms.The ability to detect and identify intracellular molecules to characterize cell accumulations was performed using the flow cytometry technique.Chalcone 7f efficiently induced cell cycle arrest at G1/S phase with percentage of 51.33% and 46.12%, respectively comparing to the untreated cells at the expense of (G2/M) phase.The DNA content in the mitotic phase G2/M was (2.55%) relative to the control sample (13.68%).In similar literature, as indicated by Guangcheng Wang et al. 77 , novel chalcones containing indole moiety clearly exhibited cell cycle arrest in G2/M in a dose-dependent manner compared to control cells in the Hepg2 cell line.At the G1 phase, CDK protein activity induced genome replication and stimulated the transition of the G1/S phase.CDKs initiated a positive feedback loop, which further enhanced CDK activities, and this forced the cell to be divided 78 .Herein, the cell cycle arrest at G1/S phase confirmed our result in the gene expression analysis section, where CDK4 was downregulated.In addition, data obtained by flow cytometry clearly outlined that chalcone 7f stimulated early apoptotic induction of colon cancer cells significantly to reach 21.38% compared to control cells (0.32%).The total apoptotic percentage in the sample treated with chalcone7f was a promising value (39.61%) relative to the untreated one (1.65%).Also, chalcone 7f exerted certain stress on colon cancer-induced necrosis of its cells (6.82%) in comparison to the control (1.12%).In addition, chalcone 7f completely degraded the genomic DNA in HCT116 cells supporting our flowcytometric results of apoptosis.As, DNA fragmentation is a late event of the apoptosis process.Molecular docking studies were done on caspase 9, XIAP, P53 cancer mutant Y220C and MDM2 proteins.Caspase 9 is an apoptotic initiator which its activation plays a critical role in the induction of apoptosis.It is an inactive monomer and its activation is triggered by dimerization 79 .This dimerization is induced viathe interaction with a small molecule at the dimer interface triggering reorientation of the activation loop 79 .XIAP is a member of the apoptotic inhibitor protein family which inhibits apoptosis process through its inhibitory effect on caspase activity 80 .The tumor suppressor p53 is inactivated in many cancers by mutation and hence its reactivation with small molecules restores the apoptosis process 81 .MDM2 is a negative regulator of the expression of P53 www.nature.com/scientificreports/theoretical results were confirmed experimentally in the gene expression and ELISA sections.At the end of this current study, all these findings were surprising and suggested that our new chalcones especially compound 7f might be an alternative promising therapeutic option, where it inhibited colorectal carcinoma through the induction of intrinsic and extrinsic apoptotic pathways of the apoptosis mediated p53 induction.

Conclusion
Novel α-cyano-indolyl chalcones incorporating 1-phenyl-3-arylpyrazole were prepared and tested against two different cancer cell lines, lung and colorectal carcinoma.We outlined in this current paper that chalcone 7f, a promising potential anticancer compound, induced human colorectal carcinoma cell death.It was found that chalcone 7f regulated apoptosis by enhancing the expression of p53 and pro-apoptotic BAX genes, while it also depressed the production of several anti-apoptotic (bcl2 and CDK4) genes.7f strongly restricted cell growth in a dose-dependent manner as clearly indicated in the ELIZA assay regarding caspases-3,-8, and -9 productions.Chalcone 7f caused apoptosis induction and G1/S cell cycle arrest in the HCT116 cell line.Chalcone 7f completely degraded the genomic DNA of HCT116.A theoretical molecular docking study supported our experimental results in which chalcone 7f interacted with caspase 9, XIAP, P53 mutant Y220C, and MDM2 with good binding energies, confirming the ability of 7f to induce p53-dependent apoptosis mechanism (intrinsic and extrinsic pathways).Thus, we suggested that chalcone 7f might be an alternative therapeutic option for colorectal carcinoma.

Experimental Chemistry
Melting points were measured with using a Stuart melting point apparatus and were uncorrected.The IR spectra were recorded using an FTIR Bruker-vector 22 spectrophotometer as KBr pellets.The 1 H and 13 C NMR spectra were recorded in DMSO-d 6 as a solvent with Varian Mercury VXR-300 NMR spectrometer operating at 300 MHz and 75 MHz, using TMS as an internal standard.Chemical shifts were reported as δ values in ppm.Mass spectra were recorded with a Shimadzu GCMS-QP-1000 EX mass spectrometer in EI (70 eV) model.The elemental analyses were performed at the Micro Analytical Centre, Cairo University.

Cytotoxic MTT assay
Source of cell lines (A549 and HCT116) A549 and HCT116 cell lines were purchased from the Company for the production of vaccines, Sera, and drugs (VACSERA), Egypt.
MTT protocol was performed to study the viability of cells after treatment with the tested compounds.The studied cell lines (A549 and HCT116) were cultured and grown in RPMI-1640 media, while HFB4 cells were reserved in DMEM-F12 media.Both media were supplemented with 10% FBS, 1% L-glutamine, and 1% antibiotic-antimycotic mixture.The cell lines were grown at conditions of 37 °C and 5% CO 2 .10 × 10 3 of A549, and HCT116 cells and the same count of normal melanocytes (HFB4) were cultured in a 96-well plate.The selected cultured cells were kept at 37°C and 5% CO 2 overnight.Then, the medium was discarded after 24 h of incubation, and 100 µL from different concentrations of the studied compounds dissolved in fresh medium without serum, with final concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 µg/mL was added.The treated cells were then incubated for 48 h.After that, 50 µL of MTT was added into each well for 4 h for the formazan crystals to be formed.Finally, 200 µl of 10% sodium dodecyl sulphate (SDS) was added to each well, and plates were kept at room temperature overnight to allow the reaction to be stopped.The optical density at 575 nm was read using a microplate multi-well reader, and the percentage of cell viability was calculated.The dose-response curve was drawn to calculate the IC 50 values for each compound using the prism GraphPad 5 program 83,84 .5 Fluorouracil, a commercial anticancer drug was used as a reference drug (positive control).Negative untreated cell control was used in this assay.

ELISA assay
The quantitative measurements of Human caspases-3, -8, and -9 concentrations in HCT116 cell culture lysates were performed using ELISA assay.The protocols were followed up based on the manuscript instructions described in the following kits; Invitrogen human Caspase-3 Elisa Kit, Catalog # KHO1091, DRG® human Caspase-8 ELISA Kit, Catalog # (EIA-4863), and DRG® human Caspase-9 ELISA Kit, Catalog # (EIA-4860)respectively.In short, the procedure described in the above protocols was as instructed; all reagents, standards, and samples were prepared.Then, 100 μL of standard or samples were added to each well and incubated at room temperature for 2 h.Afterward, 100 μL of the prepared antibody (anti-rabbit-IgG-HRP) was added to all wells for 1 h at room temperature.Subsequently, 100 μL of the prepared TMB (tetramethyl-benzidine) substrate solution was added to all wells and incubated for 15 min.Finally, 50 μL of stop solution was added to all wells to completely inactivate the effect of the enzyme.All readings were measured immediately at a wavelength of 450 nm.From the readings of the standards, a standard curve was plotted using curve fitting software.Then, the concentrations for unknown samples and controls were measured from the standard curve 87 .

Flow cytometric analysis of cell cycle
2 × 10 6 of HCT116 cells were inoculated in 60 mm Petri dishes for 24 h and then treated with compound 7f at its IC 50 concentration for 48 h.In this study, non-treated HCT116 cells were used as a negative control.After the incubation period of 48 h, HCT116 treated cells were centrifuged at 1,200 rpm at 4 °C for 10 min.After disposing of the supernatant, the cell pellet was suspended in phosphate-buffered saline (PBS) and then centrifuged at 1200 rpm for 10 min.70% cold ethanol was added overnight to allow the fixation of the cell pellet.After the step of centrifugation, the propidium iodide (PI) mixture was added to the cell pellet for 30 min at room temperature in the darkness.Subsequently, cells were subjected to Epics XL-MCL flow cytometer (BeckmanCoulter, Miami, Vol:.( 1234567890

Flow cytometric analysis of apoptosis
The percentage of apoptotic cells was detected using Annexin V-FITC kit catalogue number (#K101-25).Briefly, 5 × 10 5 of HCT116 cells were collected by centrifugation.Then, the cell pellet was suspended in 500 µL of 1X Binding Buffer.Afterward, 5 µL of Annexin V-FITC and 5 µL of propidium iodide were added to cells and kept at room temperature for 5 min in the dark [89][90][91] .Finally, the percentage of the apoptotic cells was detected by flow cytometry using the FITC signal detector(usually FL-1) and PI staining by the phycoerythrin emission signal detector (usually FL-2).

DNA fragmentation
The DNA gel electrophoresis laddering assay in the HCT116 cell line was performed according to the instructions of Yawata 87 with some modifications.Briefly, after 48 h of exposure of the HCT116 cancer cell line to compound 7f in Petri dishes (60 × 15 mm, Greiner), the cells were suspended in 1 ml of medium and then centrifuged at 900 rpm for 10 min.The genomic DNA was extracted as demonstrated in Yawata 87 .About 1 × 10 6 cells of HCT116 were treated with the IC 50 value of compound 7f.Then, the treated cells were collected via trypsinization and washed with Dulbecco's Phosphate Buffered Saline.Afterwards, the cells were treated with the lysis buffer (5 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Triton X-100, 10 mM Tris (pH 7.4), and 150 mM NaCl) for 35 min on ice.Then, the lysates were centrifuged at 10.000xg for 20 min.The degraded DNA was extracted from the supernatant with an equal volume of isoamyl alcohol: chloroform: neutral phenol mixture (1:24:25).At last, the fragmented DNA percentage was detected by performing gel electrophoresis using 2% agarose gel containing 0.1 μg/ml ethidium bromide (Supplementary File 1).

Figure 3 .
Figure 3. Cytotoxic effect of the synthesized compounds against the tested cancer cell lines (A549 and HCT116) and normal melanocytes HFB4 cells.The graphs were plotted with standard deviation (SD) using the Prism software program (Graph Pad software incorporated, version 3).

Figure 5 .
Figure 5.The schematic diagram outlined expression values of tested four genes (Bax, p53, Bcl2, and CDK4) for both control and cells treated with chalcone 7f.

Figure 6 .Figure 7 .
Figure 6.The effect of various concentrations of 7f on caspase 3 production in colorectal carcinoma.HCT116 cells were incubated in the absence (control) or presence of different concentrations of 7f, after which the caspase 3 protein level was measured by ELISA.

Figure 8 .Figure 9 .Figure 10 .Figure 11 .
Figure 8.The effect of various concentrations of 7f on caspase 8 production in colorectal carcinoma.HCT116 cells were incubated in the absence (control) or presence of different concentrations of 7f, after which the caspase 8 protein level was measured by ELISA.

Figure 14 .
Figure 14.Cell cycle analysis of chalcone 7f treated HCT116 cells relative to control HCT 116 cell line.

Figure 15 .
Figure 15.The schematic diagram illustrated the percentages of the total, early, late apoptosis, and necrosis of control and treated sample 7f.

Figure 16 .
Figure 16.The 2% Agarose gel electrophoresis diagram showed DNA samples of both untreated HCT116 cells and cells treated with 6.76 µg/mL of chalcone 7f. after 48h of treatment.

Table 1 .
IC 50 values for the prepared compounds against the selected cell lines (A549, HCT116, and HFB4), 5-Fluorouracil was used as a positive control.

Table 3 .
The values of Gibbs free energy (S) (Kcal/mol) and the types of interactions of compound 7f with the active site of caspase 9, XIAP, P53 cancer mutant Y220C, and MDM2 proteins.RMSD means root mean squared deviation.